Changes in Cementation of Reef Building Oysters Transitioning from

Letter Cite This: ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX

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Changes in Cementation of Reef Building Oysters Transitioning from Larvae to Adults Andrés M. Tibabuzo Perdomo,†,‡ Erik M. Alberts,†,‡ Stephen D. Taylor,‡ Debra M. Sherman,§ Chia-Ping Huang,§ and Jonathan J. Wilker*,‡,⊥ ‡

Department of Chemistry, Purdue University, 560 Oval Drive, West Lafayette, Indiana 47907-2084, United States Life Science Microscopy Facility, Purdue University, 170 South University Street, West Lafayette, Indiana 47907, United States ⊥ School of Materials Engineering, Purdue University, Neil Armstrong Hall of Engineering, 701 West Stadium Avenue, West Lafayette, Indiana 47907-2045, United States §

S Supporting Information *

ABSTRACT: Oysters construct extensive reef communities, providing food, protection from storms, and healthy coastlines. We still do not have a clear picture of how these animals attach to surfaces. Efforts described herein provide the first examination of adhesion at the transition from free swimming larvae to initial substrate attachment, through metamorphosis, and on to adulthood. Two different bonding systems were found to coexist. Larvae use an organic, hydrated glue that persists while the animal progresses into the juvenile phase, at which point a very different adhesive emerges. Juveniles bond with an organic−inorganic composite system, positioning the organic component for maximum adhesion by residing between the animal and substrate. Beyond understanding our marine environment, these insights may aid efforts in aquaculture, reef restoration, and adhesive design. KEYWORDS: adhesion, biomineralization, cement, oyster, reef often a flurry of media attention calling for oyster restoration to protect our coasts. Restoring marine ecosystems relies upon our ability to coax these shellfish into settling and producing glue. Conversely, aquaculturists wishing to sell single oysters instead of clusters for food and ship owners seeking minimal drag on their hulls are looking for ways to prevent cementation. Given the importance of oysters in our lives, we are compelled to understand how these shellfish stick together. Decades ago, we learned that initial larval settlement relies upon a fibrous adhesive containing proteins and mucopolysaccharides.6−10 Separate studies with the adhesives of juveniles or adults for different oyster species are somewhat contradictory, with some reports of predominantly inorganic, crystalline materials being responsible for bonding.11−14 Alternatively, the periostracum, an organic outer coating for protecting shells,15 may contribute to surface attachment.16,17 Our most recent insights on adult cement found the adhesive to be derived from organic components within an unstructured inorganic matrix.18−20 In addition to proteins and polysaccharides, the material contained phospholipids,19 possibly creating an analogy to larval barnacle adhesive.21 Both the structure and composition of adult oyster cement differed dramatically from the surrounding shell.18−20 Results discovered here show that

Oysters have been influencing human culture and our livelihood for centuries. Native Americans and European settlers in North America relied upon these shellfish to provide a major source of food.1,2 Oyster reef structures protect coasts by absorbing the energy of storm surges, creating an environment for other species to live within, and filtering large volumes of water.3,4 Once they settle onto a surface, these bivalves remain in place for their entire lives and, even after death, their shells provide a substrate for future generations. Consequently, we are particularly interested to watch how the animals transition from free swimming larvae to attached juveniles and then become macroscopic reef builders. Our current view of oysters bonding to surfaces is sparse and does not provide a consistent story. Efforts described herein examine the interface between shell and substrate while the animal develops from larvae through to adults. Oysters are shown to create two, strikingly different adhesive systems, depending upon the animal’s stage of life. Contrary to prior proposals, juvenile attachment is neither simply shell nor the periostracum shell coating. An all organic material starts and then gives way to an organic−inorganic composite system, all the while differentiating shell on top from adhesive on the bottom. Reef communities are built by oysters producing an adhesive, often called a “cement,” for sticking to one another (Figure 1A).5 As a result of fishing, pollution, and disease, a mere 2% of indigenous reef habitats remain in the US.3 Consequently, after each major storm such as hurricanes Katrina and Sandy, there is © XXXX American Chemical Society

Received: January 23, 2018 Accepted: April 13, 2018 Published: April 13, 2018 A

DOI: 10.1021/acsami.8b01305 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX

Letter

ACS Applied Materials & Interfaces

Figure 1. Adhering oysters. (A) Reef in South Carolina, US. The top animals are living and bonded to the remaining shells and cement of prior generations. (B) Two larval oysters exploring a surface. (C) Scanning electron micrograph (SEM) of an oyster after initial attachment. Note the adhesive residing between shell and surface. (D) Fluorescence microscopy of initial larval adhesive, looking up through the glass substrate at the bottom of the animal (λexcitation = 310−390 nm, λemission = 420+ nm). (E−G) SEM images from the bottom of a ∼6 month old oyster, after detachment from a plastic substrate. The adhesive has taken on a highly fibrous structure. (H−J) At 1−2 days after initial settlement, a “stage 2” adhesive, different from the larval glue, begins to emerge from in between the animal’s shells. This adhesive appears to be pliable and making efficient contact with the surface for bonding.

Looking up at the bottom of the animal through the glass substrate showed that the outer adhesive edge fluoresced (Figure 1D, Figure S1). This light emission indicated the presence of a system with reactive chemistry as opposed to, for example, the generally sticky polysaccharides used by bacteria for attachment.23 In mussels organic radicals have been shown to be involved in the curing of their protein-based adhesive.24 Radicals tend to be quite reactive and, among other things, generate radical−radical couplings.25 The resulting products are often conjugated aromatic compounds. Once these species are formed, the systems will often fluoresce.26−28 The images shown here and below are all without any added dyes. Such observations of autofluorescence indicate that these oysters form conjugated organics by way of curing chemistry that may involve radical species or other means of, for example, coupling aromatic amino acids. Energy-dispersive X-ray (EDX) spectroscopy of this early stage glue showed pronounced differences compared to the animal’s shell (Figure S2). The elements C, Cl, and S were all elevated in the adhesive whereas Ca and O were lower. Oyster shells are comprised of ∼98% inorganic CaCO3,29 but larval

oysters do not use shell or periostracum for contacting the substrate. An all organic material is responsible for larval adhesion. After metamorphosis into juveniles, a structured composite of organic and inorganic components is then generated for surface attachment. Studies began with free swimming larvae of the Eastern oyster (Crassostrea virginica), the dominant species of the US east coast and Gulf of Mexico. At less than 14 days old, larvae were grown in aquaria with glass microscope slides lining the tank bottoms. Animals were observed before, during, and post settlement. Figure 1B and Video S1 show larvae moving freely prior to settlement, consistent with a prior description.22 Within hours of contacting the substrate, an adhesive material was visible. A scanning electron micrograph (SEM) in Figure 1C showed what happened immediately after attachment, with material placed between shell and substrate. Prior studies with a different oyster species, the European flat oyster (Ostrea edulis), found initial settlement to use an unstructured organic glue.6−9 Results here for the Eastern oyster appeared to be generally similar. B


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Figure 2. Adhesion changes after metamorphosis. (A) Side-on view of an oyster growing after ∼2 months. (B) Optical microscopy of the bottom of a juvenile oyster, 5 days after settlement, looking through the glass substrate. Both stage 1 and stage 2 adhesives can be seen. (C) Different animal examined by fluorescence microscopy (λexcitation = 450−490 nm, λemission = 520+ nm), also through the glass substrate. This oyster is 4 days post settlement. (D) Two oyster spat, ∼2 months old, aggregating together in the first stages of reef construction. (E) SEM image of the whole animal and (F) a close-up of the top shell growth. Inorganic columns with gaps in between can be seen. (G) Fluorescence microscopy indicating that organics provide a binder, holding together the inorganic columns. The animals in E, F, and G are shown 7 days after settlement (λexcitation = 450− 490 nm, λemission = 520+ nm).

adhesive appeared to be more organic. The nature of this material was generally sticky, with diatoms becoming attached occasionally (Figure S3). Although larval adhesive appeared unstructured in the first few hours after deposition (Figure 1C, Figures S1−S3), a highly fibrous structure could be seen in this same material ∼6 months later. Growth on a plastic slide allowed the substrate to be bent for releasing the animal with minimal damage to the adhesive. Figure 1E−G shows a view of the bottom, formerly in contact with the surface. The fibrous structure was produced by several animals of this age (Figure S4). At 1−2 days after settlement, the animals underwent metamorphosis from a prodissoconch to a dissoconch.16 Coincident with this transition to juvenile (i.e., spat) was a dramatic change in the oyster’s adhesive system. The “stage 1” larval adhesive seen in Figure 1C−G and Figures S1−S3 did not grow any bigger. Rather, a totally new “stage 2” system appeared. Figure 1H−J shows the very first evidence of a new adhesive system emerging from in between the two shells. A pliable, organic material originated from the animal, reached the substrates, and conformed to the surfaces with an adhesive layer. Beyond the first 1−2 days, animals became larger and deposited more of this stage 2 material (Figure 2A). All subsequent growth occurred at this second adhesive, with the animal no longer adding to the stage 1 glue. Figure 2B−C shows optical and fluorescence microscopy images of juveniles less than a week after settling, viewed from the bottom of glass substrates. Both the stage 1 and stage 2 systems were seen here by SEM (Figure S5). At this point in the growth cycle, the animals aggregated by cementing to each other (Figure 2D,

Figure S6). Here, we were able to witness the earliest steps of reef formation. This stage 2 adhesive differed from stage 1 in both structure and composition. Looking down onto a growing oyster, columns with narrow gaps in between were visible (Figure 2E, F). Fluorescence microscopy showed the presence of an organic binder holding together columns of inorganic material (Figure 2G). Energy-dispersive X-ray scans across this assembly attested to the organic−inorganic composite character (Figure S7). When moving from the inorganic columns to the organic binder, Ca and C decreased while Cl and Na increased. These results indicated that the columns were predominantly CaCO3, whereas the binder was organic and hydrated with seawater. Bradford staining provided evidence that protein was present in the binder (data not shown). Overall, this assembly looked to be simply an oyster shell.30−33 However, almost every known biological or synthetic adhesive is predominantly organic. A material such as shell comprised of ∼98% CaCO329 is not likely able to generate sufficiently strong adhesive contacts. Closer inspection was required to understand how these juvenile oysters can attach in the face of intertidal forces. With oysters a week or less after settlement, examination of the newest leading edge material (Figure 3A, B) provided distinction from shell (Figure 2E−G). This new growth appeared to have the columns of shell on top. However, an amorphous layer making direct adhesive contact with the substrate was clearly visible. Fluorescence microscopy (Figure 2C) attested to the organic nature of this adhesive material and that curing chemistry was likely present. Much like the stage 1 adhesive seen in Figure 1D, autofluorescence seen here indicates that reactive chemistry, perhaps from organic radicals, is generating conjugated organic compounds. Furthermore, this C


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remained occasionally, but these structures did not look to be bound together well, and attachment to the substrate appeared tenuous (Figure 3D). By contrast, the untreated animals of Figure 3A, B seemed to be anchored more robustly. These data provide a model for juvenile oyster attachment in which a layer of organic adhesive provides an interface between the substrate and left valve (Figure 3E). The common view of shell with CaCO3 columns tied together by organics persists.30,34,35 This shell is then attached to the surface via a flowing, pliable, organic material present for maximizing interfacial contacts. The animals continued to grow larger and deposit more material. When ∼6 months old, they became big enough to allow cutting of complete cross sections (Figure 4A). Such

Figure 3. Juvenile oysters creating adhesive contacts. (A, B) Leading edge of spat, depositing new material as they grow. Inorganic columns, looking like shell, are on the top. Immediately below the inorganics are layers of amorphous material making adhesive contact with the surface. These images are from different animals, 2 days post settlement. (C) EDTA etch of a juvenile oyster for leaving behind organics after selective inorganic CaCO3 removal. Organic adhesive at the surface and the binder between CaCO3 columns persisted. (D) Partial bleach etching for selective removal of organics. This treatment often destroyed samples because of the binder and adhesive no longer being present. Here, the inorganic columns can be seen held together only loosely. See image A for a contrast of when the organics were present. (E) Model of how oysters adhere immediately after metamorphosis.

Figure 4. Adhesion of ∼6 month old oysters. (A) Animal cut into a cross-section. This picture is a composite of several SEM images. (B) Close-up of the top, thick shell with a thin periostracum coating. (C) Bottom of the animal at the surface, showing that the adhesive layer is significantly thicker than the periostracum of the top shell in frame B. (D) Interface between shell and substrate of another animal. This sample was prepared by fracturing the animal and substrate.

chemical activity did not appear to be distributed evenly across all of the new material. Rather, the fluorescence was strongest where each animal contacted the surface most recently. At ∼6 months, the leading edge of growth took on a less structured, but still familiar, appearance (Figure S8). The top, right valve (i.e., shell) was visible and distinct from the lower, left valve. At the substrate, organics were visible as well as inorganics of the lower valve. Oysters always settle with their left valve onto a substrate.16 Here we were able to observe differentiation of left versus right valves. Treating these young oysters (e.g., 1 month) with the chelating agent ethylenediaminetetraacetic acid (EDTA) removed inorganic CaCO3 selectively, leaving behind the binder between columns as well as organic adhesive atop the substrate (Figure 3C). With the exception of adhesive remaining at the surface, this image looks much like what happens with analogous chelation of shell.30,34,35 A different story emerged with bleach etching for digestion of the organics (Figure 3D, Figure S9). Most often, bleach destroyed the samples completely by removing the binder between columns and adhesive at the substrate. The persisting inorganic columns

samples permitted examination of the top, right valve in direct contrast to the surface contacting left, bottom valve. In general, the upper shells were found to be thicker, whereas the lower shells closest to the surface were thinner. The top shell was covered with a very thin periostracum coating, typically only ∼0.2 μm (Figure 4B). This organic barrier is particularly narrow for oysters relative to other shellfish.15 Another layer was observed beneath the animal, often of ∼1 μm or greater thickness. This underlayer was always more substantial than the right valve periostracum (Figure 4C). Fluorescence microscopy of spat cross sections showed strong emission in the space between shell and surface, further indicating the presence of organics providing interfacial binding (Figure S10). Looking underneath the animal here also yielded contrasts in shell, adhesive, and the substrate (Figure 4D). The crystalline, foliated sheet structure of shell33 was observed. Intermediate D



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between this shell and the surface was an adhesive layer, devoid of any conspicuous structure. With the animal being older, the adhesive was more mineralized. Further growth into adulthood was consistent with prior findings of an unstructured adhesive layer bonding shell to substrate.18−20 Results reported here indicate that the adhesive is a distinct part of the animal and not simply periostracum or shell. Regular shell plus thin periostracum of the animal’s thicker top valve differed significantly from the lower valve, which comprised a thick organic layer residing between substrate and thin shell. We can now see how oysters differentiate one side from another with regards to shells and binding atop surfaces. These observations provide our first comprehensive view of how oysters mature throughout their lives with regard to adhesion. The nature of their glue changes from initial settlement, on to juvenile adhesion, and then into adulthood. An amorphous, organic, hydrated material sticks larvae to surfaces. After metamorphosis, the animals switch over to an organic−inorganic composite adhesive system. The structure is defined by inorganic columns atop a thick, organic, underlying layer of glue. This adhesive contrasts with shell in being more organic and lacking microstructure. Growth of full-sized adults and their extensive reef structures then builds upon the materials seen here. By describing oyster adhesion through all major life stages, we hope to provide insights to those working in adhesive design, aquaculture, and reef restoration.

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Letter

ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsami.8b01305. Electron microscopy images and energy-dispersive X-ray data (PDF) Video S1, larval oysters swimming, probing the surface, and depositing glue (AVI)

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. ORCID

Jonathan J. Wilker: 0000-0002-7602-9892 Author Contributions †

A.M.T.P. and E.M.A. contributed equally to this work

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We are indebted to Karen Sisler at the Virginia Institute of Marine Sciences as well as Megan Gima and John Supan at Louisiana State University for providing larval oysters. Christopher Gilpin, Laurie Mueller, and Robert Seiler at the Purdue University Life Science Microscopy Facility helped with images. Gabriela Sincich-Sosa created the illustration for Figure 3E. This work was supported by the Office of Naval Research grants N000140710082, N000141010098, and N000141310245.

MATERIALS AND METHODS

Culturing of Oysters. About 150 000−200 000 pediveliger larvae of Eastern oysters (Crassostrea virginica) were obtained several times, over multiple spawning seasons and years from the Virginia Institute of Marine Sciences and Louisiana State University. Upon arrival in the laboratory, larvae were acclimated in aged seawater (18 parts per thousand salinity) for 15−30 min prior to transferring into an aerated 10 gallon glass aquarium at the same salinity, maintained at room temperature. Beds of plastic (vinyl, Rinzl) and glass (Thermo Scientific Gold Seal) slides were placed at the bottom of the aquaria for larval settlement. Oysters where fed daily with an algae mixture (Shellfish Diet 1800) and water changes performed every 3 days to ensure growth into spat. After 48 h, most larvae were in the crawling-settling stage (see Video S1). Instrumentation. Optical and fluorescence microscopies were carried out on an Olympus BX51 with USH-102DH and USHIO lamps as well as an Olympus DP71 CCD camera. Wavelengths for the filters used to change the excitation and emission wavelengths are provided in the figure captions. An FEI Quanta 3D FEG dual-beam scanning electron microscope (SEM) as well as an FEI Nova NanoSEM, both with EverhartThornley and through-the-lens detectors (TLD), were used. Typical parameters included 5−20 kV accelerating voltages and 4.5−10 μm working distances. Oysters were covered with platinum via a sputter coater prior to imaging. Energy-dispersive X-ray (EDX) spectroscopy was accomplished with an Oxford INCA Xstream-2 on a Quanta 3D FEG microscope. For EDX parameters, 20 kV, 50 μm objective aperture, and 100 s of collection time were used most often. Oxford AZtecEnergy EDS software was employed for data analyses. Etching. In order to differentiate between organic and inorganic materials, EDTA and bleach etching was performed. For removal of the inorganic material, a solution of 10% EDTA at pH 7.4 was used. These oysters were treated for 1 week. To remove the organics, we made diluted solutions of commercial bleach. Overnight treatment resulted in complete removal of samples from the slides. Thus, shorter incubation periods of 1 h or less were used for allowing material to persist.

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REFERENCES

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Changes in Cementation of Reef Building Oysters Transitioning from

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